The invention relates to methods for identifying genes encoding novel proteins.
There is considerable medical interest in secreted and membrane-associated mammalian proteins. Many such proteins, for example, cytokines, are important for inducing the growth or differentiation of cells with which they interact or for triggering one or more specific cellular responses.
An important goal in the design and development of new therapies is the identification and characterization of secreted proteins and the genes which encode them. Traditionally, this goal has been pursued by identifying a particular response of a particular cell type and attempting to isolate and purify a secreted protein capable of eliciting the response. This approach is limited by a number of factors. First, certain secreted proteins will not be identified because the responses they evoke may not be recognizable or measurable. Second, because in vitro assays must be used to isolate and purify secreted proteins, somewhat artificial systems must be used. This raises the possibility that certain important secreted proteins will not be identified unless the features of the in vitro system (e.g., cell line, culture medium, or growth conditions) accurately reflect the in vivo milieu. Third, the complexity of the effects of secreted proteins on the cells with which they interact vastly complicates the task of isolating important secreted proteins. Any given cell can be simultaneously subject to the effects of two or more secreted proteins. Because any two secreted proteins will not have the same effect on a given cell and because the effect of a first secreted protein on a given cell can alter the effect of a second secreted protein on the same cell, it can be difficult to isolate the secreted protein or proteins responsible for a given physiological response. In addition, certain secreted and membrane-associated proteins may be expressed at levels that are too low to detect by biological assay or protein purification.
In another approach, genes encoding secreted proteins have been isolated using DNA probes or PCR oligonucleotides which recognize sequence motifs present in genes encoding known secreted protein. In addition, homology-directed searching of Expressed Sequence Tag (EST) sequences derived by high-throughput sequencing of specific cDNA libraries has been used to identify genes encoding secreted proteins. These approaches depend for their success on a high degree of similarity between the DNA sequences used as probes and the unknown genes or EST sequences.
More recently, methods have been developed that permit the identification of cDNAs encoding a signal sequence capable of directing the secretion of a particular protein from certain cell types. Both Honjo, U.S. Pat. No. 5,525,486, and Jacobs, U.S. Pat. No. 5,536,637, describe such methods. These methods are said to be capable of identifying secreted proteins.
The demonstrated clinical utility of several secreted proteins in the treatment of human disease, for example, erythropoietin, granulocyte-macrophage colony stimulating factor (GM-CSF), human growth hormone, and various interleukins, has generated considerable interest in the identification of novel secreted proteins. The method of the invention can be employed as a tool in the discovery of such novel proteins.